Nonalcoholic fatty liver disease (NAFLD) is caused by hepatic steatosis (lipid accumulation), is a common disease that affects up to 29 million adults in the U.S., and will become the number one cause of liver disease worldwide by 2020. There are few effective ways to prevent or treat this disease. A better understanding of its etiology is needed to improve diagnosis and treatment. We previously found that NAFLD is heritable (genetically influenced) and identified common (minor allele frequency (MAF) >5%) single nucleotide polymorphisms (SNPs) at 5 loci that associate with hepatic steatosis first in ~7,000 individuals of European ancestry and then across ancestries. Variants at the 5 loci together explain ~20% of heritability suggesting that other influential variants remain to be discovered. We and others have now found that genes harboring functional coding (affecting protein sequence) common variants of small effect often can also harbor rare variants with large effects. At three of the five associated loci we have now identified putative functional coding variants across ancestries. Recent whole genome and exome sequencing studies identified many new coding variants but many are low frequency (MAF 1-5%) or rare (MAF < 1%) and neutral as opposed to damaging. These low frequency and rare variants have been underexplored in genome-wide association studies due to the limitations of imputation and small sample sizes. These variants can now be affordably assayed in large numbers of samples using the new Illumina Human Exome BeadChip. Further, we can test whether particular genes and variants affect hepatic lipid accumulation in a new cell based model of hepatic steatosis we have created. We hypothesize that low frequency (MAF 1-5%) and rare (MAF <1%) coding variants with effects on hepatic steatosis exist and if expressed in liver, affect liver lipid accumulation. To identify and characterize low and rare frequency variants for effects on NAFLD, we have now assembled the largest, most ancestrally diverse population based collection of individuals (>16,000 from 8 cohorts) with measures of hepatic steatosis and genotyped them with the Exome BeadChip. We will harmonize the hepatic steatosis phenotype and genotypes in our cohorts and carry out single variant, gene based, and conditional meta analyses across groups to identify putative causal variants. We will follow up top associating variants and genes from hepatic steatosis analyses in >4,000 histologically confirmed NAFLD cases and ~3,000 controls to replicate our findings. Using our cellular model of hepatic steatosis, we will overexpress and knockdown liver expressed genes harboring coding variants in human liver cell lines and characterize their genetic and lipidomic mechanisms of action. We will start with the three genes we have already identified that harbor coding variants that associate with NAFLD and then proceed to new genes identified and replicated from the proposed exome analyses. This work will begin to define the genetic and metabolic mechanisms which cause NAFLD to inform development of new biomarkers as well as potential therapeutics for this condition.